Administration of a selective il-6-trans-signalling inhibitor

ABSTRACT

A selective IL-6-trans-signalling inhibitor can be used to treat a variety of IL-6-mediated conditions, including inflammatory diseases and cancer. The inhibitor can safely be administered to humans at a variety of doses. Moreover, the inhibitor lessens deleterious effects associated with other IL-6 inhibitors such as lowering neutrophil counts, platelet counts and levels of C-reactive protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/532,092, filed May 31, 2017, which is the National Stage ofInternational Application No. PCT/M2015/002459, filed Dec. 1, 2015,which claims the benefit of U.S. Provisional Application No. 62/086,054,filed Dec. 1, 2014, the contents of each of which are herebyincorporated herein by reference in their entirety.

SEQUENCE LISTING

In accordance with 37 CFR § 1.52(e)(5), a Sequence Listing in the formof a text file (entitled “P110641PC00_Sequence_listing.txt”, created onMay 31, 2017, and having a size of 39,640 bytes) is hereby incorporatedby reference in its entirety.

BACKGROUND

IL-6 is a pleiotropic cytokine produced by hematopoietic andnon-hematopoietic cells, e.g. in response to infection and tissuedamage. IL-6 exerts its multiple biological activities through two mainsignalling pathways, a so-called classic ligand-receptor pathway viamembrane-bound IL-6R present mainly on hepatocytes and certainleukocytes, and a trans-signalling pathway via circulating sIL-6Roriginating from proteolytic cleavage of the membrane-bound IL-6R orfrom alternative splicing.

In the classic pathway, IL-6 directly binds to membrane-bound IL-6R onthe surface of a limited range of cell types. The IL-6/IL-6R complexassociates with a pre-formed dimer of the signal-transducing gp130receptor protein, causing steric changes in the gp130 homodimer andthereby initiating an intracellular signalling cascade. Classicsignalling is responsible for acute inflammatory defense mechanisms andcrucial physiological IL-6 functions, such as growth and regenerativesignals for intestinal epithelial cells.

The extracellular domains of IL-6R and gp130 can be generated withoutthe membrane-anchoring domains by translation of alternatively-splicedmRNAs resulting in sIL-6R and sgp130 variants. Additionally, theextracellular domain of IL-6R can be shed by membrane-bound proteases ofthe A disintegrin and metalloprotease (ADAM) family (in humans, ADAM17)to generate sIL-6R. In the trans-signalling process, sIL-6R binds toIL-6, forming an agonistic complex which binds to trans-membrane gp130dimers present on a multitude of cell types that do not expressmembrane-bound IL-6R; IL-6 signalling by signal transducers andactivators of transcription (STATs) is then induced in cells which donot normally respond to IL-6. The activity of the IL-6/sIL-6R complex isnormally controlled by levels of sgp130 present in the circulation whicheffectively compete with membrane-bound gp130. Trans-signalling ismainly involved in chronic inflammation and has been shown to preventdisease-promoting mucosal T-cell populations from going into apoptosis.

It would be desirable to have a molecule that mimics the naturaltrans-signalling inhibitor sgp130, but with a higher binding affinityand, consequently, a stronger inhibitory activity. Moreover, it would bedesirable to have a molecule that can be administered to humans withminimal toxicity and immunogenic potential.

SUMMARY OF THE INVENTION

It has now been found that a selective IL-6-trans-signalling inhibitorcan be administered to humans without any significant deleteriouseffects over a large dosage range. Moreover, it has been surprisinglyfound that the terminal half-life of the inhibitor allows dosing on aweekly, biweekly (i.e., every other week), monthly or even lesserfrequency.

In certain embodiments, the invention includes an inhibitor (e.g., apolypeptide dimer as disclosed herein) for the treatment of aninflammatory disease or IL-6-mediated condition, wherein the polypeptideis administered at a dose of 0.5 mg to 5 g. The invention also includesa method of treating inflammatory disease by administering the inhibitor(e.g., a polypeptide dimer as disclosed herein), where the inhibitordose is from 0.5 mg to 5 g. The invention further includes use of suchan inhibitor in the manufacture of a medicament for treating aninflammatory disease at the indicated dose. Preferably, a human istreated.

In other embodiments, the invention includes a polypeptide dimer asdisclosed herein for treating an IL-6-mediated condition withoutsignificantly lowering neutrophil counts, platelet counts and/or levelsof C-reactive protein or without lowering neutrophil counts, plateletcounts and/or levels of C-reactive protein below a normal range inhealthy subjects or patients suffering from an IL-6-mediated condition.The invention also includes a method of treating an IL-6-mediatedcondition by administering a polypeptide dimer as disclosed herein,wherein the method does not significantly lower neutrophil counts,platelet counts and/or levels of C-reactive protein. The inventionfurther includes use of such a polypeptide dimer in the manufacture of amedicament for treating an IL-6-mediated condition without significantlylowering neutrophil counts, platelet counts and/or levels of C-reactiveprotein. Preferably, a human is treated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the trans-signalling pathway of IL-6. sIL-6R generated fromalternatively spliced mRNA or proteolytic cleavage is able to bind toIL-6 to form a IL-6/sIL-6 complex that binds to gp130 present on thevast majority of body cell types and induce a intracellular signallingcascade.

FIG. 2 shows that a polypeptide dimer comprising two monomers of SEQ IDNO: 1 does not interfere with IL-6 binding to membrane-bound IL-6R(classic signalling), but selectively binds to the IL-6/sIL-6R complexand prevents trans-signalling.

FIG. 3 shows profiles after i.v. infusion of Peptide 1 (left panel) at0.75 mg, 7.5 mg, 75 mg, 150 mg, 300 mg, 600 mg and 750 mg and s.c.injection (right panel) at 60 mg (2×2 mL).

FIG. 4 shows profiles after intravenous administration at 75 mg, 300 mgand 750 mg in healthy subjects (left panel) and CD patients in clinicalremission (right panel).

FIG. 5 shows profiles after intravenous administration at 75 mg, 300 mgand 750 mg once a week for 4 weeks in healthy subjects.

FIG. 6 shows model predictions using a 2-compartment structural PK model(solid line) and observed data (circles) in trial 000115.

FIGS. 7A-7C show the nucleotide (SEQ ID NO: 8) and amino acid (SEQ IDNO: 9) sequence of the single gp130-Fc subunit.

FIGS. 8A-8F show nucleotide sequence elements of the expression plasmidpFER02. FIG. 8A depicts CMV IE Promoter (SEQ ID NO: 10). FIG. 8B depictsHuman IgH PolyA (SEQ ID NO: 11). FIG. 8C depicts Amp (bla) gene (SEQ IDNO: 12). FIG. 8D depicts SV40 Promoter (SEQ ID NO: 13). FIG. 8E depictsDihydrofolate Reductase Coding Sequence (SEQ ID NO: 14). FIG. 8F depictsSV40 Poly (SEQ ID NO: 15).

DETAILED DESCRIPTION OF THE INVENTION

Preferred inhibitors of the invention include a dimer of two gp130-Fcfusion monomers (e.g., two monomers of SEQ ID NO:1). In its active form,the polypeptide of SEQ ID NO: 1 exists as a dimer linked by twodisulfide linkages at Cys623 and Cys626 (FIG. 2). SEQ ID NO: 2corresponds to the amino acid sequence of a gp130-Fc fusion monomerhaving the endogenous signal peptide. The signal peptide is removedduring protein synthesis, resulting in the production of the polypeptideof SEQ ID NO: 1.

The polypeptide dimers described herein selectively inhibit excessivetrans-signalling (FIG. 1) and induces apoptosis of the detrimentalT-cells involved in multiple inflammatory diseases. The polypeptidedimer targets and neutralises IL-6/sIL-6R complexes and is thereforeexpected to only inhibit IL-6 trans-signalling in the desiredtherapeutic concentrations, leaving classic signalling and its manyphysiological functions, as well as its acute inflammatory defencemechanisms, intact (FIG. 2). The polypeptide dimer is believed to beunable to interfere with classic IL-6 signalling due to sterichindrance; the Fc portion is unable to insert into a cell membrane,making the gp130 portion unavailable for binding to membrane-boundIL-6/sIL-6R complex. Thus, the polypeptide dimer is expected to haveefficacy similar to global IL-6 blockade (e.g., tocilizumab, sirukumab)but with fewer side effects.

Polypeptide dimers described herein preferably comprise gp130-Fcmonomers having the sequence corresponding to SEQ ID NO:1. In certainembodiments, the monomers have the sequence corresponding to SEQ IDNO:2. In certain embodiments, polypeptide dimers described hereincomprise polypeptides having at least 90%, 95%, 97%, 98%, 99% or 99.5%sequence identity to SEQ ID NO: 1 or SEQ ID NO:2. Preferably, thepolypeptide comprises the gp130 D6 domain (in particular amino acidsTFTTPKFAQGE: amino acid positions 585-595 of SEQ ID NO:1), AEGA in theFc domain hinge region (amino acid positions 609-612 of SEQ ID NO:1) anddoes not comprise a linker between the gp130 portion and the Fc domain.In a preferred embodiment, the disclosure provides a polypeptide dimercomprising two monomers having an amino acid sequence at least 90%sequence identify to SEQ ID NO: 1, wherein the amino acid sequencecomprises the gp130 D6 domain, AEGA in the Fc domain hinge region, andthere is no linker present between the gp130 portion and the Fc domain.In a preferred embodiment, the disclosure provides a polypeptide dimercomprising two monomers having an amino acid sequence at least 90%sequence identify to SEQ ID NO: 2, wherein the amino acid sequencecomprises the gp130 D6 domain, AEGA in the Fc domain hinge region, andthere is no linker present between the gp130 portion and the Fc domain.

It is desirable for polypeptides to be substantially free ofgalactose-alpha-1,3-galactose moieties, as these are associated with animmunogenic response. It was surprisingly found that dimers of theinvention have low levels of such moieties. In preferred embodiments,the polypeptide dimer contains no greater than 6% ofgalactose-alpha-1,3-galactose per mole polypeptide. Preferably, thepolypeptide dimer contains no greater than 4 mole %, 3 mole %, 2 mole %,1 mole %, 0.5 mole %, 0.2 mole %, 0.1 mole % or even an undetectablelevel of galactose-alpha-1,3-galactose (e.g., as measured by WAX-HPLC,NP-HPLC or WAX, preferably as determined by WAX-HPLC). In otherembodiments, the polypeptide dimer contains less than 6%, 4%, 3%, 2%,1%, 0.5%, 0.2%, or even 0.1% of galactose-alpha-1,3-galactose, relativeto the total amount of glycans, either by mass or on a molar basis.

It is also desirable for a polypeptide of the invention to besialylated. This has the advantage of increasing the half-life ofpolypeptides of the invention. Each chain of the polypeptide dimercontains 10 N-glycosylation sites; nine N-glycosylation sites arelocated in the gp130 portion and one N-glycosylation site is located inthe Fc portion. The polypeptide therefore contains a total of 20glycosylation sites. In certain embodiments, a mean of at least 52% orat least 54% of glycans on the polypeptide include a sialic acidresidue, such as a mean from 52-65% (e.g., as measured by WAX-HPLC,NP-HPLC or WAX, preferably as determined by WAX-HPLC). Preferably, thepolypeptide of the invention has an approximate molecular weight of 220kDa; each 93 kDA having an additional ˜20 kDa molecular weight derivedfrom 10 N-glycosylation chains.

It is further desirable to minimize the extent to which polypeptidesaggregate, which is herein referred to as oligomerization which resultsin oligomeric aggregates. “Oligomeric aggregates” as used herein, doesnot refer to the active dimerized peptide. Instead, the term refers toat least a dimer of active dimers. In was surprisingly found that thepeptide dimers of the invention display low levels of aggregation. Incertain embodiments, less than 5%, less than 4%, less than 3%, less than2%, less than 1.5%, or even less than 1.0% of the polypeptide is presentas an oligomer. The oligomer content can be measured, for example, bysize exclusion chromatography-multi angle light scatting (SEC-MALS) orSEC-UV.

Preferably, the polypeptide dimer is present in its full-length form(e.g., includes two full length monomers, e.g., of SEQ ID NO:1).However, cell culture can produce a truncated variant referred to hereinas the single gp130 form (SGF). SGF is a covalently-bound two-chainmolecule, one chain comprising a full-length gp130-Fc monomer (e.g., ofSEQ ID NO:1) and a second chain comprising a truncated gp130-Fc monomer(e.g., a truncation of SEQ ID NO:1), which second chain includes the Fcdomain and lacks most or all of the gp130 domain (e.g., terminatedbefore the linker sequence to the Fc region). Studies to datedemonstrate that SGF does not have a heterogeneous amino-terminus. SGFcan be formed at consistent levels in a bioreactor and once formed, SGFlevels are not readily changed during purification, processing oraccelerated storage conditions. SGF levels can be difficult to removeduring purification due to similar physical-chemical properties to thefull-length form of the polypeptide dimer; thus efforts to remove SGFcan result in a significant reduction in yield. It was surprisinglyfound that polypeptide dimers of the invention are nearly alwaysfull-length. In certain embodiments, the composition of the inventioncomprises polypeptide dimers comprising no greater than 4.0% by weight,3.0% by weight, 2.0% by weight or even 1.5% by weight of polypeptidesthat are a truncated variation of the polypeptide of SEQ ID NO: 1 withrespect to polypeptides of SEQ ID NO: 1. In certain embodiments, thecomposition of the invention comprises no greater than 4.0% by weight,3.0% by weight, 2.0% by weight or even 1.5% by weight of polypeptidesthat are a truncated variation of the polypeptide of SEQ ID NO: 2 withrespect to polypeptides of SEQ ID NO: 2.

Dosing

The doses described herein represent a dose range that are believed tobe safe and tolerable, based upon Phase I data. Other compoundstargeting IL-6R or IL-6 have often displayed, in early clinical trials,decreased neutrophil and platelet counts and lower levels of C-reactiveprotein (CRP) both in healthy subjects and patients with RA. However,the observed levels in neutrophil and platelet counts in healthysubjects dosed with the polypeptide of the invention were still withinthe normal range. It appears, from the results from the Phase I program,that the polypeptide of the invention does not display the same effectson biomarkers as compounds targeting IL-6R or IL-6.

In the two trials with the polypeptide dimers comprising monomers of SEQID NO: 1, an ex vivo assay measuring the level of activation of STAT 3by stimulating whole blood samples from the subjects with hyper IL-6 wasemployed as an assessment of the activity of the drug. Concentrationlevels of the polypeptide comprising monomers of SEQ ID NO: 1 above 1μg/mL are believed to be related to suppressed signal to baseline in thesecondary messenger (STAT3) assay. The concentration level of thepolypeptide dimers comprising monomers of SEQ ID NO: 1 would correspondto the peak levels of the 7.5 mg dose. A dose of 75 mg administered asan i.v. infusion has been shown to have a concentration above 1 μg/mL atsteady state for a dosing interval of one week. The corresponding doseadministered every two weeks is 300 mg. It is believed that 60 mgadministered as a subcutaneous injection is believed to result in thesame steady state for dosing every week.

In certain embodiments, the dose is from 0.5 mg to 5 g polypeptidedimer. For example, the dose can be from 5 mg to 3 g, 10 mg to 2 g, 60mg to 1 g or preferably from 60 mg to 750 mg.

The polypeptide dimers can be administered at a frequency appropriatefor the intended condition. In certain embodiments, the polypeptidedimer is dosed once every 7-60 days. For example, the polypeptide dimerscan be dosed once every 7-30 days or 7-20 days. In preferredembodiments, the dose occurs weekly (once every 7 days) or biweekly(once every 14 days). Doses can also occur on a daily basis or twice- orthrice-weekly. A dose refers to a single dosing episode, whether thedose is a unit dosage form or multiple unit dosage forms taken together(e.g., ingestion of two or more pills, receiving two or moreinjections). As discussed below, this dose frequency could not bepredicted from animal studies. Human clinical trials found a meanhalf-life of 4.6 days to 5.5 days. In contrast, cynomolgus monkeys had ahalf-life of only 0.7 days when administered the polypeptide dimersintravenously and 1.4-1.5 days subcutaneously.

The polypeptide dimer of the invention is typically administeredparenterally, such as intravenously or subcutaneously. Administrationcan occur according to one of the dosing frequencies disclosed herein.

In certain embodiments, the polypeptide dimer is administeredintravenously, dosed once every 7-60 days with a dose from 60 mg to 1 g.

In certain such embodiments, the polypeptide dimer is administeredintravenously, dosed once every 7-30 days with a dose from 60 mg to 1 g.

In an exemplary embodiment, the polypeptide dimer is administeredintravenously, dosed weekly with a dose from 60 mg to 1 g.

In another exemplary embodiment, the polypeptide dimer is administeredintravenously, dosed biweekly with a dose from 60 mg to 1 g.

In certain embodiments, the polypeptide dimer is administeredsubcutaneously, dosed once every 7-60 days with a dose from 60 mg to 600g.

In certain embodiments, the polypeptide dimer is administeredsubcutaneously, dosed once every 7-30 days with a dose from 60 mg to 600g.

In an exemplary embodiment, the polypeptide dimer is administeredsubcutaneously, dosed once weekly with a dose from 60 mg to 600 g.

In another exemplary embodiment, the polypeptide dimer is administeredsubcutaneously, dosed once biweekly with a dose from 60 mg to 600 g.

Safety

The polypeptide dimer comprising monomers of SEQ ID NO: 1 has beenadministered up to 750 mg as a single dose and 600 mg once weekly for 4weeks. The safety profile of the polypeptide was favourable with fewadverse events occurring in all treatment groups, including the placebogroup, all being mild or moderate. No apparent dose-related trends inincidence or frequency of adverse events were observed. There were noapparent dose-related trends or treatment related changes in vitalsigns, ECG, or clinical chemistry parameters. Three events of infusionreactions occurred, all were mild/moderate with cutaneous symptoms likeurticaria and swelling, and rapidly resolved without any sequelae.

Overall, the polypeptide dimer comprising monomers of SEQ ID NO: 1 wassafe and well tolerated when administered i.v. up to 600 mg once weeklyfor 4 weeks and up to 750 mg as a single dose.

The potential risk of the polypeptide comprising monomers of SEQ ID NO:1 in humans can also be addressed indirectly by analysing the clinicalstudies investigating similar compounds targeting IL-6R or IL-6. Todate, there is no approved compound which blocks the same signallingpathway as this polypeptide dimer, i.e. targeting and neutralisingIL-6/sIL-6R-complex to inhibit the trans-signalling pathway, without anyinteraction with either IL-6 or IL-6R individually. However, there areexperiences with compounds targeting IL-6 receptors. One of thesecompound is tocilizumab, which has been approved in Europe and UnitedStates. Tocilizumab binds specifically to both soluble andmembrane-bound IL-6 receptors and has been shown to inhibit sIL-6R andmIL-6R mediated signalling.

The most common reported adverse drug reactions in RA patients treatedwith tocilizumab (occurring in ≥5%) were upper respiratory tractinfections, nasopharyngitis, headache, hypertension and increased ALT.The most serious adverse drug reactions were serious infections,complications of diverticulits and hypersensitivity reactions. Decreasesin neutrophil and platelet counts have occurred following treatment withtocilizumab. Decreases in neutrophil counts below 10⁹/L occurred in 3.4%of patients on tocilizumab 8 mg/kg plus disease-modifying anti-rheumaticdrugs (DMARDs). Approximately half of the patients who developed an ANC<10⁹/L did so within 8 weeks after starting therapy. Decreases below5×10⁸/L were reported in 0.3% patients receiving tocilizumab 8 mg/kg andDMARDs. Severe neutropenia may be associated with an increased risk ofserious infections, although there has been no clear association betweendecreases in neutrophils and the occurrence of serious infections inclinical trials with tocilizumab to date. Events reported during theinfusion were primarily episodes of hypertension; events reported within24 hours of finishing an infusion were headache and skin reactions(rash, urticaria). These events were not treatment limiting. Clinicallysignificant hypersensitivity reactions associated with tocilizumab andrequiring treatment discontinuation were reported in a total of 13 outof 3,778 patients (0.3%) treated with tocilizumab during the controlledand open-label clinical studies. These reactions were generally observedduring the second to fifth infusions of tocilizumab. Gastrointestinalperforations, primarily in patients with a history of diverticulitis,have been reported as rare events, both in tocilizumab clinical trialsand post-marketing. The etiology is unclear but RA patients have agenerally increased risk for perforations of both the upper and lower GItract (regardless of DMARD therapy); the risk is highest in RA patientson glucocorticoid therapy, NSAIDs, or with a history of diverticulitis.Fatal anaphylaxis has been reported after marketing authorisation duringtreatment with tocilizumab. It should be noted that RA patients may haveother background diseases as confounding factors.

Because the polypeptide dimer comprising monomers of SEQ ID NO: 1 is afirst-in-class fusion protein, comparisons with the different monoclonalantibody products with different mechanisms of action is of limitedvalue. In contrast to other products which completely block IL-6activity, this polypeptide dimer is believed to only interfere with theIL-6/sIL-6R complex, leaving the membrane bound IL-6 pathway accessible.

IL-6 has a broad involvement in the immune and inflammatory responses inthe body. When both soluble and membrane-bound IL-6R is blocked, thereis potentially an increased risk of infections and otherimmuno-dependent diseases as well as a less prominent inflammatoryresponse. While not wishing to be bound by theory, it is believed thattreatment with the polypeptide dimer comprising monomers of SEQ ID NO:1, which only targets the IL-6/sIL-6R complex, would prevent theperpetuation of chronic intestinal inflammation in IBD and preserve theacute phase inflammatory response activated by classical IL-6signalling, thereby lowering the risk of opportunistic infections.However, it could not be predicted whether there is a concentration ofpolypeptide dimer of the invention above which classical IL-6 signallingwould be impacted. Thus, the data herein surprisingly demonstrate thatthere is less impact on classical IL-6 signalling relative to othertreatments targeting IL-6 activity.

Based on the data presented herein, an advantage of the polypeptidedimer of the invention is that it may have a lesser effect on neutrophilcounts, platelet counts and/or levels of C-reactive protein than othercompounds that inhibit IL-6. In certain embodiments, the polypeptidedimer of the invention does not significantly lower neutrophil counts,platelet counts and/or levels of C-reactive protein or without loweringneutrophil counts, platelet counts and/or levels of C-reactive proteinbelow a normal range in healthy subjects or patients suffering from anIL-6-mediated condition. For example, the administration of thepolypeptide dimer at a dose amount described herein maintains neutrophilcounts, platelet counts and/or levels of C-reactive protein within anormal physiological range. In certain embodiments, neutrophil counts,platelet counts and/or levels of C-reactive protein are no more than50%, 40%, 30%, 20%, 15%, 10% or 5% less than the lower limit of thenormal physiological range. The measurement of neutrophil counts,platelet counts and/or levels of C-reactive protein can occurimmediately after treatment, one day after days, three days aftertreatment, one week after treatment, two weeks after treatment, onemonth after treatment, three months after treatment, six months aftertreatment or a year after treatment.

The determination of neutrophil counts, platelet counts, and levels ofC-reactive protein can be performed by any number of assays well-knownin the art. Neutrophil count, also referred to as absolute neutrophilcount (ANC) is a measure of the number of neutrophil granulocytespresent in blood (see, e.g., Al-Gwaiz LA, Babay HH (2007). “Thediagnostic value of absolute neutrophil count, band count andmorphologic changes of neutrophils in predicting bacterial infections”.Med Princ Pract 16 (5): 344-7). Normal physiological values forC-reactive protein in adult males and females are 0-5.00 mg/L (e.g., viaturbidimetry). Normal physiological values for neutrophils in adultfemales are 1.61-6.45×10⁹ per L (absolute value, e.g., via laser flowcytometry) or 37.9-70.5% (calculated); in adult males, the correspondingvalues are 1.46-5.85×10⁹ per L and 38.2-71.5%. Normal physiologicalvalues for platelets in adult females are 173-369×10⁹ per L (e.g., viahigh frequency impedance measurement); in adult males, the correspondingvalues are 155-342×10⁹ per L.

The polypeptide dimer of the invention preferably does not significantlyinduce the formation of antibodies (e.g., antibodies to the polypeptidedimer) in humans. Even more preferably, the antibodies are notneutralizing antibodies. In certain embodiments, antibodies against thepolypeptide dimer of the invention are detectable in fewer than 5%, 2%,1%, 0.5%, 0.2%, 0.1% or 0.01% of treated subjects or patients.Typically, the limit of detection is approximately 9 ng/mL serum.

Indications

In acute inflammation, IL-6 has been shown to induce the acute phaseresponse in the liver leading to release of the cascade of acute phaseproteins, in particular CRP. By forming a complex with sIL-6R shed byapoptotic neutrophils at the site of inflammation and binding of theresulting IL-6/sIL-6R trans-signalling complex to the signal transducergp130 on endothelial cells, IL-6 induces expression of chemokines suchas monocyte chemotactic protein (MCP)-1 and attracts mononuclear cells.This leads to the resolution of acute inflammation and to the initiationof an adaptive immune response. Thus, in acute inflammation, IL-6 withsIL-6R complex supports the transition between the early predominantlyneutrophilic stage of inflammation and the more sustained mononuclearcell influx ultimately also leading to the resolution of inflammation.

Chronic inflammation, such as in Crohn's disease (CD), ulcerativecolitis (UC), rheumatoid arthritis (RA) or psoriasis, is histologicallyassociated with the presence of mononuclear cells, such as macrophagesand lymphocytes, persisting in the tissue after having been acquired forthe resolution of the acute inflammatory phase. In models of chronicinflammatory diseases, IL-6 seems to have a detrimental role favouringmononuclear-cell accumulation at the site of injury, through inductionof continuous MCP-1 secretion, angio-proliferation and anti-apoptoticfunctions on T-cells.

Inflammatory bowel disease (IBD), namely CD or UC, is a chronicinflammation occurring in the gut of susceptible individuals that isbelieved to be independent of a specific pathogen. Alterations in theepithelial mucosal barrier with increased intestinal permeability leadto an enhanced exposure of the mucosal immune system to luminalantigens, which causes an inappropriate activation of the intestinalimmune system in patients. The uncontrolled activation of mucosal CD4+T-lymphocytes with the consecutive excessive release of proinflammatorycytokines induces pathogenic gastrointestinal inflammation and tissuedamage. There is a consensus that the main activated immune cellsinvolved in the pathogenesis of IBD are intestinal T-cells andmacrophages.

IL-6 is shown to be a central cytokine in IBD in humans. Patients withCD and UC have been found to produce increased levels of IL-6 whencompared with controls, the IL-6 levels being correlated to clinicalactivity. CD patients have also been found to have increased levels ofsIL-6R and consequently, IL-6/sIL-6R complex in serum. Lamina propriamononuclear cells obtained from surgical colon specimens from patientswith CD and UC showed that both CD4+ T-cells and macrophages producedincreased amounts of IL-6 compared to controls. sIL-6R was found to bereleased via shedding from the surface of macrophages and mononuclearcells with increased production associated with elevated levels of IL-6.In patients with CD, mucosal T-cells showed strong evidence for IL-6trans-signalling with activation of STAT3, bcl-2 and bcl-xl. Theblockade of IL-6 trans-signalling caused T-cell apoptosis, indicatingthat the IL-6/sIL-6R system mediates the resistance of T-cells toapoptosis in CD.

Thus, in IBD patients, acquired accumulation of disease-promoting CD4+T-cells in the lamina propria leading to perpetuation of inflammation iscritically dependent on anti-apoptotic IL-6/sIL-6R trans-signalling. Itis believed that by acting on the IL-6/sIL-6R complex, the polypeptidedimer disclosed herein is useful in treating CD and other inflammatorydiseases.

Thus, the polypeptide dimer of the invention can treat IL-6-mediatedconditions. IL-6-mediated conditions include inflammatory disease or acancer. In this regard, the polypeptides and compositions describedherein may be administered to a subject having an inflammatory disease,such as juvenile idiopathic arthritis, Crohn's disease, colitis (e.g.,colitis not associated with IBD, including radiation colitis,diverticular colitis, ischemic colitis, infectious colitis, celiacdisease, autoimmune colitis, or colitis resulting from allergiesaffecting the colon), dermatitis, psoriasis, uveitis, diverticulitis,hepatitis, irritable bowel syndrome (IBS), lupus erythematous,nephritis, Parkinson's disease, ulcerative colitis, multiple sclerosis(MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma, andvarious cardiovascular diseases such as atherosclerosis and vasculitis.In certain embodiments, the inflammatory disease is selected from thegroup consisting of, diabetes, gout, cryopyrin-associated periodicsyndrome, and chronic obstructive pulmonary disorder.

Preferably, the inflammatory disease or IL-6-mediated condition isinflammatory bowel disease, preferably wherein the treatment induces theremission of inflammatory bowel disease. Preferably, the inflammatorybowel disease is Crohn's disease or ulcerative colitis, preferablywherein the treatment maintains the remission of inflammatory boweldisease. Preferably, the inflammatory disease or IL-6-mediated conditionis rheumatoid arthritis, psoriasis, uveitis or atherosclerosis.Preferably, the inflammatory disease or IL-6-mediated condition iscolitis not associated with inflammatory bowel disease, preferablywherein the colitis is radiation colitis, diverticular colitis, ischemiccolitis, infectious colitis, celiac disease, autoimmune colitis, orcolitis resulting from allergies affecting the colon.

For inflammatory disease such as inflammatory bowel disease, treatmentcan include remission of the condition, maintenance of remission of thecondition, or both.

Other embodiments provide a method of treating, reducing the severity ofor preventing a cancer, including, but not limited to multiple myeloma,plasma cell leukemia, renal cell carcinoma, Kaposi's sarcoma, colorectalcancer, gastric cancer, melanoma, leukemia, lymphoma, glioma,glioblastoma multiforme, lung cancer (including but not limited tonon-small cell lung cancer (NSCLC; both adenocarcinoma and squamous cellcarcinoma)), non-Hodgkin's lymphoma, Hodgkin's disease, plasmocytoma,sarcoma, thymoma, breast cancer, prostate cancer, hepatocellularcarcinoma, bladder cancer, uterine cancer, pancreatic cancer, esophagealcancer, brain cancer, head and neck cancers, ovarian cancer, cervicalcancer, testicular cancer, stomach cancer, esophageal cancer, hepatoma,acute lymphoblastic leukemia (ALL), T-ALL, acute myelogenous leukemia(AML), chronic myelogenous leukemia (CIVIL), and chronic lymphocyticleukemia (CLL), salivary carcinomas, or other cancers.

Further embodiments of the present disclosure provide a method oftreating, reducing the severity of or preventing a disease selected fromthe group consisting of sepsis, bone resorption (osteoporosis),cachexia, cancer-related fatigue, psoriasis, systemic-onset juvenileidiopathic arthritis, systemic lupus erythematosus (SLE), mesangialproliferative glomerulonephritis, hyper gammaglobulinemia, Castleman'sdisease, IgM gammopathy, cardiac myxoma and autoimmune insulin-dependentdiabetes.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

The polypeptide dimer of the invention can be administered inconjunction with a second active agent. The second active agent can beone or more of 5-aminosalicylic acid, azathioprine, 5-mercaptopurine anda corticosteroid. Dosage regimes for the administration of5-aminosalicylic acid, azathioprine, 5-mercaptopurine andcorticosteroids are well-known to a skilled person.

The polypeptide dimers may be produced, for example, by expressing themonomers, e.g. monomers comprising SEQ ID NO: 1, in cells. In anexemplary embodiment, a vector comprising a nucleic acid encoding SEQ IDNO: 1 or SEQ ID NO:2 is transfected into cells. The design of theexpression vector, including the selection of regulatory sequences, maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and so forth. Regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from retroviral LTRs, cytomegalovirus(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (suchas the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus majorlate promoter (AdMLP)), polyoma and strong mammalian promoters such asnative immunoglobulin and actin promoters. The host cell may be amammalian, insect, plant, bacterial, or yeast cell, preferably the cellis a mammalian cell such as a CHO cell.

The transfected cells are cultured to allow the cells to express thedesired protein. The cells and culture media are then collected andpolypeptide dimers are purified, e.g., by chromatography column steps(e.g., MAbSelect Sure, SP Sepharose, Capto Q). The dimer can also beconcentrated and/or treated with viral reduction/inactivation steps. Theresulting dimers can then be used to prepare compositions, preferablypharmaceutical compositions useful for therapy.

EXEMPLIFICATION Example 1 Animal Studies Example 1a MousePharmacokinetics

Four groups with 54 mice (27 male and 27 female) weighing 25-38 greceived a single dose of the polypeptide of SEQ ID NO: 1 in its activedimerized form (“Peptide 1”) by either i.v. (3 mg/animal) or s.c. (0.3,3 and 30 mg/animal) injection.

Bioavailability was approximately 60%, and apparent dose linearity wasobserved for AUC, AUC_(t) and C_(max). The t_(max) of 8-24 hours was asexpected for a protein. Peptide 1 was cleared slowly from the systemiccirculation with a clearance of 142 mL/day/kg. Distribution volumesestimated by the elimination phase (Vz) and first moment curve (Vss)were 397 mL/kg and 284 mL/kg, respectively, indicating that Peptide 1was distributed outside the vascular bed. The terminal half-life rangedfrom 1.3-2.3 days.

Example 1b Rat Pharmacokinetics Single-Dose Administration

The single-dose PK of Peptide 1 was investigated after i.v. and s.c.administration in two different strains of rats, Sprague Dawley (8rats/group) and Wistar (24 rats/group), showing somewhat differentresults. The clearance (57 and 93 mL/kg/day, respectively) anddistribution volume appeared to be lower in the Sprague Dawley rat, witha 2-fold higher bioavailability, 60%, as compared to approximately 30%for the Wistar rat. T_(max) was observed 0.5-1.5 days after s.c.administration, and the terminal half-life was approximately 2 days,ranging from 1.7-2.7 days, with only small differences between the twoadministration routes.

Repeat-Dose Administration

Intravenous Administration

Rats (n=18) received i.v. bolus doses 10, 30 and 100 mg/kg/occasiontwice a week for 2 weeks. The increase in C_(max) and AUC after thefirst administration seemed to be approximately dose linear. However,exposure appeared to be consistently higher in males than in females atall dose levels. The 100 mg/kg/occasion group reached C_(max) levels of2100 μg/mL for male rats and 1740 μg/mL for female rats. The AUC_(t)values were 942 and 642 day×μg/mL.

After two weeks, the systemic exposure to Peptide 1 in rats decreasedfor the 10 mg/kg and 30 mg/kg dose groups. As anti-drug antibody (ADA)responses were confirmed in all animals by Week 8 of the study,decreases in expected exposure over time may be attributed toADA-mediated clearance of Peptide 1.

Subcutaneous Administration

In 2- and 4-week repeat dose s.c. studies, the C_(max) and AUC after thefirst administration seemed to increase approximately dose linearly.However, at the last dose, a notably lower drug exposure was seen,possibly due to antibody formation; all animals tested showed antibodiestowards Peptide 1.

Example 1c Cynomolgus Monkey Pharmacokinetics Single-Dose Administration

The PK of single administration of Peptide 1 was investigated in maleand female cynomolgus monkeys at doses of 0.1-100 mg/kg s.c. (n=4) and1.0 mg/kg i.v (n=4). The bioavailability was approximately 60% followings.c. administration with a t_(max) of 6-24 hours. The clearance was 98mL/day/kg, and the distribution volume approximately 70-90 mL/kg. Thehalf-life determined after i.v. administration was 0.68 days, andapproximately 1.5 days after s.c. administration.

Repeat-Dose Administration

Cynomolgus monkeys were dosed s.c. with 2 (n=4), 10 (n=4), 50 (n=2), or100 (n=2) mg/kg Peptide 1 twice weekly for 2 weeks, and with 10 (n=4),30 (n=4), or 100 (n=4) mg/kg Peptide 1 twice weekly for 4 weeks. Theexposure by means of C_(max) and AUC increased approximately doselinearly, and similar exposure and maximal concentration between thefirst and last dose at the higher doses were observed after 2 weeks.However, after 4 weeks, the last administered dose showed a lower drugexposure compared to the first administration, possibly due to antibodyformation. The mean half-life of Peptide 1 ranged from 1.0-1.8 days forthe first administration in the different treatment groups with ashorter half-life after the last administration.

Example 2 Clinical Trial 000067 (Single Dose) Design

This was a single-dose, placebo controlled, single blinded, randomisedwithin dose, parallel group dose-escalating trial. The trial wasconducted in two parts, where Part 1 included healthy subjects and Part2 included patients with CD in clinical remission. The objective was toexamine the safety and tolerability, and if possible, to obtain signs ofpharmacological effects, after single doses of Peptide 1.

In Part 1, 64 subjects were included, of whom 48 (44 men, 4 women)received active treatment and 16 (all men) received placebo. Seven doseswere investigated and administered as an i.v. infusion over 30 minutes(0.75 mg, 7.5 mg, 75 mg), or 1 hour (150 mg, 300 mg, 600 mg, and 750mg). In addition, 6 subjects received a s.c. dose of 60 mg Peptide 1 and2 subjects received a s.c. dose of placebo. Peptide 1 was administeredat 15 mg/mL in 25 mM histidine, 200 mM sucrose and 0.1 mg/mL polysorbate20.

In Part 2, 24 patients were included, of whom 18 (11 men, 7 women),received active treatment (75 mg, 300 mg, and 750 mg) and 6 (4 men, 2women) received placebo, all administered by i.v.

Results

The PK evaluation after i.v. administrations of Peptide 1 showed doseproportionality for both AUC and Cmax in the range 0.75 mg to 750 mg,the Cmax concentrations in plasma ranging from 0.2 to 170 μg/mL (FIG.3). The clearance was approx. 0.13 L/h, the mean terminal half-lifeapprox. 4.5 days, and the distribution volume approx. 20 L, the latterindicating some extravascular distribution. The s.c. administration of60 mg Peptide 1 showed a Cmax of 1.1 μg/mL at 2.3 days, and a half-lifeof 5.0 days. The bioavailability after s.c. administration of Peptide 1was calculated to be approx. 50%. There was no indication oftarget-mediated drug disposition.

The i.v. administration of 75, 300, and 750 mg to CD patients inremission showed very similar results as for the healthy subjects (FIG.4). The AUC and Cmax were dose proportional with Cmax concentrations of16, 76, and 186 μg/mL (16, 77, and 161 μg/mL for healthy subjects). Theclearance was approx. 0.13 L/h, the mean terminal half-life approx. 4.6days, and the distribution volume approx. 22 L.

The safety profile of Peptide 1 was favourable with few adverse eventsoccurring in all treatment groups, including the placebo group, allbeing mild or moderate. No apparent dose-related trends in incidence orfrequency of adverse events were observed. The infusions werediscontinued in two subjects, one due to mild (Part 1, 300 mg group) andone due to moderate (Part 2, 75 mg group) infusion reactions.

There were no apparent dose-related trends or treatment-related changesin vital signs, ECG, or clinical chemistry parameters (includingneutrophil counts, platelet counts, or C-reactive protein levels).

One healthy subject in the 300 mg group showed non-neutralisingtreatment emergent anti-Peptide 1 antibodies at the follow-up visit 5-6weeks after administration.

Overall, Peptide 1 was safe and well tolerated when administeredintravenously up to 750 mg as a single i.v. dose, and at 60 mg as asingle s.c. dose.

Example 3 Clinical Trial 000115 (Multiple Ascending Dose) Design

This was a placebo controlled, double-blind, within dose-grouprandomised, parallel group trial with the objective to investigate thesafety, tolerability, and pharmacokinetics of multiple ascending dosesof Peptide 1. The doses investigated were 75, 300 and 600 mg Peptide 1administered once a week, for 4 weeks, by i.v. infusion over 30 minutes(75 mg) or 1 hour (300 mg and 600 mg).

Twenty-four (24) healthy subjects were included, of whom 18 (11 men and7 women) received active treatment and 6 (2 men and 4 women) receivedplacebo.

Results

The PK evaluation showed very close characteristics on the first andlast treatment days, and similar to the results in the single-dosestudy. The AUC and Cmax were dose proportional after first and fourthdosing with Cmax concentrations of 19, 78, and 148 μg/mL after the firstdose, and 19, 79, and 142 μg/mL after the fourth dose (16, 77, and 161μg/mL for single dose in healthy subjects; FIG. 5). The correspondingtrough values were 0.66, 2.68, 4.56 μg/mL and 0.98, 3.95 and 7.67 μg/mLfor the three dose levels. The mean terminal half-life as calculatedafter the last dose was approx. 5.5 days.

The safety profile of Peptide 1 was favourable with few adverse eventsoccurring in all treatment groups, including the placebo group, allbeing mild or moderate. No apparent dose-related trends in incidence orfrequency of adverse events were observed. One subject (600 mg group)was withdrawn due to mild infusion reactions.

There were no apparent dose-related trends or treatment related changesin vital signs, ECG, or clinical chemistry parameters (includingneutrophil counts, platelet counts, or C-reactive protein levels).

No anti-Peptide 1 antibodies were detected in any of the subjects.

Overall, Peptide 1 was safe and well tolerated when administered i.v. upto 600 mg once weekly for 4 weeks.

Example 4 Modeling of Pharmacokinetic Data

The PK data from the 000115 trial can be adequately described using a2-compartment structural model. Predicted profiles of 75, 300 and 600 mgof Peptide 1 and observed data are depicted in FIG. 6 and the estimatedmean PK parameters are listed in Table 1.

TABLE 1 Model Estimates for Peptide 1 Using 2-compartment StructuralPharmacokinetic Model Parameter Estimate SE CV % V 1.7 L 0.08 4.8 V2 8.8L 0.32 3.6 CL 3.2 L/day 0.06 1.8 CL2 16.4 L/day 0.24 14.4

Peptide 1 has a binding affinity in humans of 130 pM to the IL-6/sIL-6Rcomplex. At doses of 75-600 mg, the occupancy level are more than 90% atestimated steady state levels of Peptide 1 using the binding affinity(KD; 130 pM) and the IL-6/sIL-6R levels (C_(target); 2.0 nM based onsIL-6R).

Example 5 Preparation of Peptide 1 Cloning and Expression of Peptide 1in CHO/dhfr—Cells

CHO/dhfr⁻ cells were obtained from the European collection of cellcultures (ECACC, No. 9406067). The adherent CHO/dhfr⁻ cells aredeficient in dihydrofolate reductase (DHFR), an enzyme that catalysesthe reduction of folate to dihydrofolate and then to tetrahydrofolate.CHO/dhfr⁻ cells thus display sensitivity to the antifolate drug,methotrexate (MTX).

The CHO/dhfr⁻ cell line is well characterised and tested. The safety ofthe CHO/dhfr⁻ parental cell line as a cell substrate for the productionof biopharmaceuticals for human use was confirmed by ECACC (Porton Down,UK) for microbial sterility, mycoplasma, and adventitious virusesaccording to 21 CFR.

Selection and Construction of the cDNA Sequence

The cDNA sequence of a monomer of Peptide 1 (the polypeptide sequence ofSEQ ID NO: 1) was synthesised as a single DNA fragment by GeneArt AG(Regensburg, Germany) using the sequence for the extracellular domain ofgp130 (IL6ST, NCBI Gene ID 3572, transcript variant 1 (NP 002175), aminoacids 23-617) and Fc domain of human IgG1 (IGHG1, NCBI Gene ID 3500,amino acids 221-447 according to Kabat EU numbering). The sequence wasoptimised for optimal codon usage in CHO cells. Three well-characterisedpoint mutations were introduced into the lower hinge region of the Fcpart.

The cDNA sequence was further modified by replacing the original gp130signal peptide with a mouse IgG heavy chain signal peptide of knownefficacy in CHO cell expression systems. The signal peptide is cleavedoff during protein synthesis. The presence of the IgG1 Cys-Pro-Pro-Cyssequence in the Fc region results in the dimerisation of two identicalgp130-Fc subunits via the sulfhydryl residues on the Fc region, whichtogether form Peptide 1.

FIG. 7 presents the nucleotide and amino acid sequence of the gp130-Fcsubunit used for the formation of Peptide 1.

Construction of the Expression Plasmid for Selection of the Master CellBank (MCB)

The monomer cDNA was cloned into a pANTVhG1 expression vector (Antitope)containing the dhfr gene for transfectant selection with MTX as follows:First, the expression vector was digested with MluI and EagI restrictionenzymes to permit the insertion of Peptide 1 cDNA. Second, the monomercoding region was PCR amplified using the OL1425 and OL1426 primers(Table 2) and digested with MluI and EagI restriction enzymes. Third,the digested fragments were gel purified and ligated together togenerate the pFER02 expression vector. The monomer cDNA was insertedunder the control of the cytomegalovirus (CMV) promoter.

Table 3 presents the function of the pFER02 expression elements. FIG. 8presents the nucleotide sequences of the pFER02 expression elements.

TABLE 2 Oligonucleotide Sequences Used to Amplify the Monomer CodingRegion for Cloning into pANTVhG1 Primer Sequence (5′-3′)* OL1425ctgttgctacgcgtgtccactccGAGCTGCTGGATCCTTGCGGC (SEQ ID NO: 6) OL1426gcgggggcttgccggccgtggcactcaCTTGCCAGGAGACAGAGACAG (SEQ ID NO: 7) *Monomer1-specific sequences are shown in upper case, vector-specific sequencesare shown in lower case and restriction sites are underlined

TABLE 3 pFER02Expression Elements Feature Function CMV promoterImmediate-early promoter/enhancer. Permits efficient, high-levelexpression of the recombinant protein hIgG1 polyA Human IgGpolyadenylation sequence Ampicillin resistance Selection of vector in E.coli gene (β-lactamase) SV40 early promoter Allows efficient, high-levelexpression and origin of the neomycin resistance gene and episomalreplication in cells expressing SV40 large T antigen DHFR Selection ofstable transfectants in CHO dhfr- cells SV40 polyadenylation Efficienttranscription termination and signal polyadenylation of mRNA

Cell Line Selection Process Leading to the Final Peptide 1 ProducingClone

The pFER02 vector was linearised with the blunt-end restriction enzymeSspI, which has a single recognition site located in the beta-lactamasegene. The linearised plasmid was transfected into 5×10⁶CHO/dhfr⁻ cellsusing lipid-mediated transfection. Twenty-four hours after transfection,transfected cells were selected in medium supplemented with 5% dialysedfoetal calf serum (FCS) and 100 nM methotrexate (MTX). Transfected cellswere diluted into this medium at various densities and dispensed into96-well, flat bottom tissue culture plates. Cells were then incubated ina humidified atmosphere at 5% CO₂ and 37° C. Fresh MTX selection mediumwas added at regular intervals during the incubation time to ensure thatMTX levels and nutrient levels remained constant.

Initial Cell Line Selection with MTX selection

For several weeks post transfection, tissue culture plates were examinedusing a Genetix CloneSelect® Imager, and >2,000 wells were observed tohave actively growing colonies. Supernatants from these wells weresampled and assayed for Peptide 1 titre by ELISA. Based on the resultsof this assay, a total of 105 of the best expressing wells were expandedinto 48-well plates. A total of 83 cell lines were selected forexpansion into 6-well plates or T-25 flasks; supernatant from each ofthe cell lines was sampled and assayed for Peptide 1 titre (ELISA).Based on these results, 54 of the best expressing cell lines withoptimal growth characteristics were selected for expansion into T-75 orT-175 flasks; supernatants from the confluent flasks were sampled andPeptide 1 titres quantified (ELISA). Comparison of the expression levelsbetween the cell lines allowed for the identification of the 38 bestcell lines which were selected for productivity analysis. Productivitywas assessed as follows:

Productivity (pg/cell/day)=((Th−Ti)/((Vh+Vi)/2))/time

Where:

-   -   Th is the harvest titre [μg/mL]    -   Ti is the initial titre [μg/mL]    -   Vh is the viable cell count at harvest [×10⁶ cells/mL]    -   Vi is the initial viable cell count [×10⁶ cells/mL]    -   Time is the elapsed time (days) between Ti and Th        Based on productivity results (pg/cell/day), 13 cell lines were        selected for gene amplification.

MTX-Driven Gene Amplification for Peptide 1 Cell Line Selection

The 13 selected cell lines were chosen for the first round of geneamplification by selective pressure under increasing concentrations ofMTX (0.1-50 M). After 7-10 days, supernatant from each well from each ofthe 13 cell lines were sampled and assayed for Peptide 1 titre (ELISA).Wells from each cell line with high Peptide 1 expression levels wereassessed for productivity (pg/cell/day). A second round of geneamplification was initiated with a total of 16 wells from cell linesthat showed significant increases in productivity.

The second round of gene amplification was conducted in the presence ofincreased MTX concentrations; supernatants from each culture wereassayed for Peptide 1 titre (ELISA). Selected wells from each cell linewere expanded and productivity was assessed (pg/cell/day); five celllines with increased productivity in response to increased MTX selectionpressure were identified. These five cell lines were progressed to athird round of gene amplification using selection pressure underincreased MTX concentration; supernatants from each well were assayedfor Peptide 1 titre (ELISA). Selected wells for each cell line wereexpanded and productivity (pg/cell/day) was assessed; five cell linesdemonstrating high Peptide 1 expression were selected.

Limiting Dilution of Clones

Limiting dilution cloning was performed on the five cell linesdemonstrating Peptide 1 expression. After one week of incubation, plateswere examined using a Genetix CloneSelect® Imager and single colonieswere identified. The growth rates of two cell lines during dilutioncloning were noted as being particularly slow and so these cell lineswere discontinued. In total, from the three remaining cell lines, 58clonal colonies were selected for expansion, first into 48-well platesand then successively expanded through 12-well plates, T-25 flasks andT-75 flasks in the absence of MTX. Each of the 58 selected clones wasthen assessed for productivity (pg/cell/day); 16 clones were selectedfor suspension adaptation and adaptation to growth in achemically-defined medium.

Adaptation of Cell Lines to Suspension Culture in Chemically DefinedMedium

The 16 cell lines were adapted to suspension culture in achemically-defined medium as follows: selected cell lines in adherentculture were first adapted to suspension both in CHO suspension growthmedium (DMEM high glucose, including L-glutamine and sodium pyruvate, 5%dialysed FCS, 20 mg/L L-proline, 1× penicillin/streptomycin, 1% pluronicF68) and then in chemically defined suspension growth medium (CDOpti-CHO® from Life Technologies Ltd. (Paisley, UK), 2.5% dialysed FCS,0.1× penicillin/streptomycin, 8 mM Glutamax®).

Once adapted to suspension culture, the cell lines were weaned, instages, into a serum-free chemically-defined suspension growth medium(CD Opti-CHO®, 0.1× penicillin/streptomycin, 8 mM Glutamax®). MTX wasomitted from all suspension cultures. The adapted lines were expandedand seed cell banks were prepared. Briefly, cells were expanded to 300mL total volume and harvested when cell density exceeded 0.85×10⁶cells/mL and viability was >90%. A further 3×10⁷ cells were seeded intoa fresh flask containing 70 mL suspension growth medium for growth andproductivity analysis. The remaining cells were harvested bycentrifugation and resuspended in an appropriate volume of freezingmedium to yield a cell suspension at 1×10⁷ cells/mL. Vials were frozendown to −80° C. The cell bank was then transferred to liquid nitrogenfor long-term storage.

The 16 cell lines were further refined down to 5 clones after serum-freeadaptation. The 5 clones were assessed for growth (cell density and celldoubling time) and productivity (pg/cell/day), after which 3 clones wereselected. One clone was selected to make a master cell bank.

Preparation of the master cell bank (MCB) and working cell bank (WCB)was carried out. One vial from the pre-seed stock was used for thepreparation of a 200 vial MCB, and one vial of MCB was used to prepare a200 vial WCB. In each case, a vial was thawed and the cryopreservationmedium removed by centrifugation. The cells were resuspended andpropagated in volume in growth medium (CD OptiCHO®/4 mM L-glutamine).Four passages were performed during the creation of MCB and six passageswere performed during the creation of WCB.

When sufficient cells were obtained, cells were aliquoted incryopreservation medium (92.5% CD OptiCHO®/7.5% DMSO) into polypropylenevials (each containing approximately 1.5×10⁷ viable cells) andcryopreserved by reducing the temperature to −100° C. over a period ofat least 60 minutes in a gradual freezing process. Vials are stored in avapour phase liquid nitrogen autofill container in a GMP controlledarea.

Description of the Drug Substance (DS) Manufacturing Process

A brief description of the Peptide 1 DS manufacturing process is asfollows. Cells from a WCB vial are revived and progressively expandedusing protein-free medium prior to inoculation into a productionbioreactor. Upon completion of the cell culture, cells and cell debrisare removed by filtration of the culture.

Purification consists of three chromatography column steps (MAbSelectSure, SP Sepharose, Capto Q or Sartobind Phenyl), a concentration anddiafiltration step and includes two specific viralreduction/inactivation steps; Triton X-100 (inactivation of envelopedviruses) treatment and a nanofiltration step (removal of enveloped andnon-enveloped viruses).

Following concentration and diafiltration, excipients are added for theformulation of the DS. The formulated Peptide 1 is 0.22 μm filtered intocontainers.

Description and Composition of the Drug Product (DP)

The DP is a sterile solution to be administered by i.v. infusion. The DPconsists of Peptide 1 at a concentration of 15 mg/mL in an isotonicsolution containing 25 mM L-histidine, 200 mM sucrose and 0.1 mgpolysorbate 20/mL at pH 7.6. The vials are overlaid with nitrogen forprotection against oxidation. The product is intended for single use andstorage at −20° C. until thawing for clinical administration.

Composition and Batch Formula

The batch formula for the drug product is presented in Table 4.

TABLE 4 DP Batch Composition Component Amount Quality standard Peptide 1720 g Ferring specification L-Histidine 186.18 g Ph. Eur./USP* Sucrose3286.08 g Ph. Eur./USP* Polysorbate 20 4.8 g Ph. Eur./USP* WFI ad 49536g Ph. Eur./USP* Sodium hydroxide quantum satis Ph. Eur./USP* Nitrogenquantum satis Ph. Eur./USP* *curr. Ed.

1.-23. (canceled)
 24. A method for the treatment of an inflammatorydisease or an IL-6-mediated condition in a human, said method comprisingadministering to a human in need thereof an effective amount of apolypeptide dimer that inhibits IL-6 trans-signaling, wherein thepolypeptide dimer comprises two monomers, each monomer comprises anamino acid sequence having at least 90% sequence identity to SEQ ID NO:1, wherein the effective amount is 0.5 mg to 5 g of the polypeptidedimer.
 25. The method of claim 24, wherein each monomer comprises anamino acid sequence having at least 95% sequence identity to SEQ IDNO:
 1. 26. The method of claim 24, wherein the monomers have SEQ IDNO:
 1. 27. The method of claim 24, wherein the effective amount is 7.5mg to 1 g of the polypeptide dimer.
 28. The method of claim 24, whereinthe effective amount is 60 mg to 1 g of the polypeptide dimer.
 29. Themethod of claim 24, wherein the polypeptide dimer is administered dailyor twice- or thrice-weekly.
 30. The method of claim 24, wherein thepolypeptide dimer is administered every 7-60 days.
 31. The method ofclaim 24, wherein the polypeptide dimer is administered every 7-30 days.32. The method of claim 24, wherein the polypeptide dimer isadministered every 7-20 days.
 33. The method of claim 24, wherein thepolypeptide dimer is administered every 7 days.
 34. The method of claim31, wherein the polypeptide dimer is administered every 14 days.
 35. Themethod of claim 24, wherein the polypeptide dimer is administeredparenterally, intravenously or subcutaneously.
 36. The method of claim24, wherein the inflammatory disease or IL-6-mediated condition isinflammatory bowel disease.
 37. The method of claim 36, wherein theinflammatory bowel disease is Crohn's disease or ulcerative colitis. 38.The method of claim 36, wherein the treatment induces or maintains theremission of inflammatory bowel disease.
 39. The method of claim 24,wherein the inflammatory disease or IL-6-mediated condition isrheumatoid arthritis, psoriasis, uveitis or atherosclerosis.
 40. Themethod of claim 24, wherein the inflammatory disease or IL-6-mediatedcondition is colitis not associated with inflammatory bowel disease. 41.The method of claim 40, wherein the colitis is radiation colitis,diverticular colitis, ischemic colitis, infectious colitis, celiacdisease, autoimmune colitis, or colitis resulting from allergiesaffecting the colon.
 42. The method of claim 24, wherein neutrophilcounts, platelet counts and/or levels of C-reactive protein aremaintained within a physiologically normal range after administration ofthe polypeptide dimer.
 43. The method of claim 24, wherein each monomercomprises the gp130 D6 domain corresponding to the amino acids atpositions 585-595 of SEQ ID NO: 1, and an Fc domain hinge regioncomprising the amino acids at positions 609-612 of SEQ ID NO: 1, andeach monomer does not comprise a linker between the gp130 D6 domain andthe Fc domain hinge region.